PROJECT SUMMARY Maintaining protein homeostasis is essential for human health. Cells make millions of proteins every minute. Every step of protein biosynthesis is closely monitored by quality control mechanisms to eliminate aberrant intermediates that fail to mature. Quality control pathways triggered by ribosomes that stall aberrantly while synthesizing new proteins are major contributors to numerous human diseases including neurodegeneration, blood disorders, and various cancers. These stalls may result from mutated, damaged or misprocessed messenger RNAs and translational factors, as well as cellular stresses such as nutrient deprivation or pathogen invasion. Advances in high-throughput technologies have generated growing parts lists of cotranslational processes, global profiles of gene expression, and potential connections between different cellular pathways. Yet our understanding of how stalled ribosomes actually activate specific quality control pathways lags much behind. In particular, how cells distinguish different aberrantly stalled ribosomes from actively translating or transiently paused ribosomes remains a fundamental question in cell biology. A major limitation to answering this question is the inability to distinguish and mechanistically dissect specific ribosomal complexes from heterogeneous cellular populations. Here, I propose to systematically establish a translational arrest quality control code that identifies the specific fates of stalled nascent proteins, mRNAs, translational factors, and ribosomes in response to different types of translational stresses. To achieve this, we will use a cell-free system to reconstitute and isolate ribosomes stalled by distinct mechanisms for direct comparative analyses. We will employ a multidisciplinary approach integrating biochemistry, structural biology, proteomics, and genomics to identify specific characteristics of different types of translational arrests. We will test the utility of this quality control code to dissect the molecular mechanisms associated with two physiological processes that rely on ribosome-associated quality control: translational regulation in differentiating red blood cells and an autoregulatory mechanism that regulates the expression of tubulin mRNA. This will provide an unprecedented resource to identify and break down these important cellular pathways into individual steps for mechanistic and structural analyses. These insights will reveal general paradigms of quality control target selection and will be crucial for developing tools to investigate these complex physiologic processes in vivo and identifying new therapeutic strategies.